1. Field of the Invention
This invention relates to novel multibinding compounds (agents) that are macrolide antibiotics, aminoglycosides, lincosamides, oxazolidinones, streptogramins, tetracyclines or other compounds which bind to bacterial ribosomal RNA or one or more proteins involved in ribosomal protein synthesis in the bacterium, and to pharmaceutical compositions comprising such compounds. The compounds are useful as antibacterial agents for treating a variety of bacterial infections.
2. State of the Art
Organisms generate polypeptides (proteins) in order to survive. Organisms that cannot generate proteins cannot maintain viability. Because the majority of genes encode proteins, “gene expression” is nearly synonymous with protein synthesis. Gene expression involves two steps—transcription and translation. Genes code for proteins using various codons (units of three nucleotides), such as start codons (which initiate translation), stop codons (which stop translation) and codons in between the start and stop codons which selectively code for the various amino acids.
Translation is the RNA directed synthesis of polypeptides. This process requires all three classes of RNA. The template for correct addition of individual amino acids is the mRNA, yet both tRNAs and rRNAs are involved in the process.
Prokaryotes and eukaryotes use ribosomes to generate proteins. Ribosomes are cytoplasmic organelles, and are large complexes of proteins and three (prokaryotes) or four (eukaryotes) rRNA (ribosomal ribonucleic acid) molecules called subunits made in the nucleolus. Ribosomes serve as the site of mRNA translation. Once the two (large and small) subunits are joined by the mRNA from the nucleus, the ribosome translates the mRNA into a specific sequence of amino acids, or a polypeptide chain.
In its inactive state, the ribosome exists as two subunits; a large subunit and a small subunit. When the small subunit encounters an mRNA, the process of translation of the mRNA to protein begins. There are two sites in the large subunit, for subsequent amino acid-charged tRNAs to bind to and thus be close enough to each other for the formation of a peptide bond. The A site accepts a new tRNA bearing an amino acid, and the P site bears the tRNA attached to the growing chain.
tRNA (transfer RNA) is a specific RNA molecule which acts as a translator between mRNA and protein. Each tRNA has a specific anticodon and acceptor site. Each tRNA also has a specific charger protein; this protein can only bind to that particular tRNA and attach the correct amino acid to the acceptor site. The energy to make this bond comes from ATP. These charger proteins are called aminoacyl tRNA synthetases The tRNAs carry activated amino acids into the ribosome. The ribosome is associated with the mRNA ensuring correct access of activated tRNAs and containing the necessary enzymatic activities to catalyze peptide bond formation.
Protein synthesis proceeds from the N-terminus to the C-terminus of the protein. The ribosomes “read” the mRNA in the 5′ to 3′ direction. Active translation occurs on polyribosomes (also termed polysomes). This means that more than one ribosome can be bound to and translate a given mRNA at any one time. Chain elongation occurs by sequential addition of amino acids to the C-terminal end of the ribosome bound polypeptide. Translation proceeds in an ordered process. First accurate and efficient initiation occurs, then chain elongation and finally accurate and efficient termination must occur. All three of these processes require specific proteins, some of which are ribosome associated and some of which are separate from the ribosome, but may be temporarily associated with it.
Initiation of Translation
Before translation occurs, a ribosome must dissociate into its 30S and 50S subunits. A ternary complex termed the preinitiation complex is formed consisting of the initiator, GTP, eIF-2 and the 40S subunit. The mRNA is bound to the preinitiation complex.
Initiation of translation in both prokaryotes and eukaryotes requires a specific initiator tRNA (which includes methionine). The initiation of translation requires recognition of a start (AUG) codon.
Peptide bond formation is catalyzed by a 23S rRNA component of the 50S subunit (peptidyl transferase). Another important site for protein synthesis is the 16S rRNA component of the 30S subunit (the aminoacyl tRNA site). When protein synthesis is terminated, release factor proteins bind to the stop codons, GTP hydrolysis occurs, and peptidyl transferase activity is stimulated, causing release of the protein from the tRNA.
Elongation of the New Protein
After the first charged tRNA appears in the A site, the ribosome shifts so that the tRNA is in the P site. New charged tRNAs, corresponding to the codons of the mRNA, enter the A site, and a peptide bond is formed between the two amino acids. The first tRNA is now released and the ribosome shifts again so that a tRNA carrying two amino acids is now in the P site, and a new charged tRNA can bind to the A site. This process of elongation continues until the ribosome reaches a stop codon.
Elongation requires specific non-ribosomal proteins. Elongation of polypeptides occurs in a cyclic manner. At the end of one complete round of amino acid addition the A site will be empty and ready to accept the incoming aminoacyl-tRNA dictated by the next codon of the mRNA. This means that not only does the incoming amino acid need to be attached to the peptide chain but the ribosome must move down the mRNA to the next codon. Each incoming aminoacyl-tRNA is brought to the ribosome by an eEF-1a-GTP complex. When the correct tRNA is deposited into the A site the GTP is hydrolyzed and the eEF-1a-GDP complex dissociates. For additional translocation events to occur the GDP must be exchanged for GTP. This is carried out by eEF-1bg similarly to the GTP exchange that occurs with eIF-2 catalyzed by eIF-2B. The peptide attached to the tRNA in the P site is transferred to the amino group at the aminoacyl-tRNA in the A site. This reaction is catalyzed by peptidyltransferase in a process termed transpeptidation. The elongated peptide now resides on a tRNA in the A site. The A site needs to be freed in order to accept the next aminoacyl-tRNA. The process of moving the peptidyl-tRNA from the A site to the P site is termed, translocation. Translocation is catalyzed by eEF-2 coupled to GTP hydrolysis. In translocation, the ribosome is moved along the mRNA such that the next codon of the mRNA resides under the A site. Following translocation, eEF-2 is released from the ribosome and the cycle can start over again.
Termination of the Protein
When the ribosome reaches a stop codon, no aminoacyl tRNA binds to the empty A site. This signals the ribosomes to break into its large and small subunits, releasing the new protein and the mRNA. The protein may then undergo post-translational modifications. For example, it might be cleaved by a proteolytic (protein-cutting) enzyme at a specific place, have some of its amino acids altered, or become phosphorylated or glycosylated.
Protein Synthesis Inhibitors
In bacteria, if the ribosomal RNA is inactivated, for example, through binding of a ligand to the ribosomal RNA, protein synthesis is adversely affected and the bacteria will likely die. Many of the antibiotics, in particular, MLS antibiotics, used to treat bacterial infections function by inhibiting translation. Inhibition can be effected at all stages of translation, from initiation to elongation to termination. Many antibiotics are believed to primarily attach to the ribosomal RNA at the 16S and 23S ribosomal subunits and inhibit growth of bacteria by inhibiting protein synthesis.
Chloramphenicol inhibits prokaryotic peptidyl transferase. Streptomycin and neomycin inhibit prokaryotic peptide chain initiation, also induce mRNA misreading. Tetracycline inhibits prokaryotic aminoacyl-tRNA binding to the ribosome small subunit. Erythromycin inhibits prokaryotic translocation through the ribosome large subunit, and fusidic acid functions in a manner similar to erythromycin.
MLS antibiotics such as erythromycin have been used for years to treat various infections, for example, those caused by gram positive bacteria, gram negative bacteria and anaerobic bacteria. Many bacteria are becoming drug resistant. One theory of how the bacteria become drug resistant is that their ribosomal RNA mutates such that the antibiotics no longer bind as ligands to the RNA.
A number of macrolide antibiotics are orally bioavailable, but in contact with the gastrointestinal tract, degrade to some degree to form products which cause adverse side effects such as diarrhea. For this reason, many derivatives of naturally occurring macrolide antibiotics such as erythromycin are esterified at the 6-position to minimize formation of the degradation products following oral administration.
It would be advantageous to provide agents useful as antibiotics which have higher bioavailability, and are less subject to degradation and which have improved affinity for ribosomal RNA. The present invention provides such agents.